Every time my cells remains 50% after changing the media followed by gentle wash with PBS, and just particles surround in the media after overnight incubation in 5% CO2 incubator.
Hi Nabeel
What transfection reagent are you using to KD your cells? Many transfection reagents are highly toxic to cells and hence some protocols recommend to change the media after transfecting the cells well within 4-6 hrs of transfection because effects like you mentioned are very commonly observed.
The toxicity is sometimes cryptic and seen in situations while changing the media or cleaning them with PBS. Here are some recommendations
1) If you are using 10% serum instead transfect the cells in reduced serum condition (2%) like in Opti MEM Reduced Serum Media
2)Additionally do not try to transfect too much quantity of whatever you are transfecting. If there is high concentration of the genetic material then you might want to switch to electroporation rather than lipofection. The concentration of transfection reagent and nucleic acid is critical to maximizing transfection efficiency while minimizing cellular toxicity.
3)How about % confluency when you are transfecting the cells. I hope it is not more than 70% by any means.
4)Also check for the general health of your cells whether they are in good health while you are normally passaging them.
5) How about passage number. Higher the passage number, lower is the transfection efficiency and high passage number renders cells towards altered morphology, genetic constitution etc.
Hope it helps!!!
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